ABSTRACT/SUMMARY Wnt proteins are phylogenetically conserved, secreted glycoproteins that regulate cell-cell communication during development and adult tissue homeostasis. Wnt binding to both Frizzled, which has structural similarities to G-protein coupled receptors, and to the co-receptors Lrp5/6, initiates canonical (i.e. Wnt/b- catenin) signaling, representing the most widely studied type of Wnt signaling. We and others demonstrated that the intestinal epithelium is an archetypal canonical Wnt/b-catenin-dependent tissue, with a final common phenotype of loss of intestinal crypts ? and crucially ? loss of Lgr5+ intestinal stem cells (ISC) uniformly apparent upon by inhibition of Wnt ligands, their production, receptors or signaling. Because of this critical role in the intestine and other stem cell compartments, the Wnt signaling axis is an important therapeutic target, but unfavorable biochemical properties have impeded the use of Wnt as a regenerative drug. We determined the first binary crystal structure of a Wnt in complex with a Frizzled ligand binding domain (CRD)? a breakthrough that offered diverse insights into Wnt function. This revealed the molecular basis for Wnt/Fz cross-reactivity, and elucidated the Wnt lipidation site and its essential role in Fz binding (Science, 2012). This application exploits a synergistic collaboration between Chris Garcia and Calvin Kuo at Stanford (Multi-PIs) to molecularly characterize Wnt/receptor interactions through biophysical imaging approaches, thus enabling the structure-based protein engineering of artificial bioengineered Wnt analogs which are directly applied to mechanistic and therapeutic investigation of the intestinal epithelium and Lgr5+ ISC. Aim 1 continues our structural efforts to image the entire Wnt/Frizzled/Lrp6 ternary transmembrane complex by X-ray crystallography and cryo-Electron Microscopy, building on preliminary successes in expressing and purifying this multimolecular assembly. Aim 2 focuses on a potentially transformative new discovery we have made that overcomes two major obstacles for translation of Wnts into therapeutics: 1- difficulty of expressing natural Wnts as recombinant proteins due to their lipidation and 2- Fz cross-reactivity. We have developed water-soluble, Fz-specific surrogate Wnt agonists that mimic all aspects of Wnt activity but as an easily expressed non-lipidated recombinant protein. These engineered surrogate Wnt agonists are not only biochemically tractable gain- and loss-of-function probes for basic Wnt signaling mechanisms, but offer a new strategy for exploiting the Wnt signaling axis in regenerative medicine and will be structurally optimized in Aim 2 for specific activity, Fz specificity and intestinal organoid growth. Lastly, Aim 3 explores the in vivo potential of these bioengineered surrogates for support of Lgr5+ intestinal stem cells, mitigation of intestinal radiation damage and augmented transplantation of human intestinal organoids. Collectively, this proposal pursues basic molecular insights into Wnt structure and signal initiation mechanisms, and applies this information to practical problems in Wnt research and intestinal biology. !